Apparatus for making chemical separations



Oct. 6, 1964 R. w. ALLINGTON APPARATUS FOR MAKING CHEMICAL SEPARATIONS Filed Oct. 23, 1961 3 Sheets-Sheet l URN 71481 E 20 Den/E Mame oemewsr w nuasauacro su qy OFDENSt'A/aU/fl em 7 2 m w m i .M ew m M 6 70 7a a a Y M m a 5N p 5 5 M L T INVENTOR. ROBERT M ALLINGTON' ATTORNEYS Oct. 6, 1964 R. w. ALLINGTON APPARATUS FOR MAKING CHEMICAL SEPARATIONS Filed Oct. 25, 1961 3 Sheets-Sheet 2 United States Patent 3,151,639 APPARATUS FQR lvlAKiNG CEMECAL SEEARATEGNS This invention relates to apparatus for making chemical separations and more particularly to apparatus for transferring fractioned material from a tubular container into separate test tubes, which material may be a mixture of macromolecules within a density gradient centrifuge tube or light absorbing solut s migrating through a chromatographic coliunn.

Density gradient centrifugation is accomplished by placing a liquid possessing an axial gradient of density in a centrifuge tube. The gradient may be formed by pipetting a layer of concentrated, dense, solute, in solution, into the bottom of the centrifuge tube. Solutions of sucrose or cesium chloride are commonly used for this purpose. Onto the dense layer are pipetted additional layers of progressively less concentrated solutions thereby forming a density gradient which is maintained in a metastable state by the force or" gravity. Once the gradient column has been so formed, a mixture of macromolecules is pipetted onto the surface of the column. When the tube containing the density gradient column is spun in a centrifuge, the macromolecules begin to sediment. As a molecule of a given species tends to sediment faster than others of the same species, by reason of random factors such as diffusion, it will encounter a region of higher density in the density gradient column, which wih tend to slow down the movement of the molecule. Conversely, if the particular molecule lags behind the molecules or" the same species, its movement will tend to speed up. In consequence, the diiierent molecules will migrate in discrete zones down the density gradient column, each species of molecule usually migrating at a different rate. When the centrifuge is stopped, the macromolecules will have formed into discrete, transverse zones in the tube.

The main diiliculty associated with the density gradient technique has been the problem of how to remove and/or quantitatively analyze the various zones within the tube. Attempts have been made to remove the zones with a hypodermic needle, either through the side of the tube or down from the top of the tube. This method suffers from two practical defects, namely, the difficulty of distinguishing the various zones by eye and the turbulence which accompanies the removal of one zone and tends to destroy the remaining zones. Another technique proposed is to puncture the bottom of the tube and collect the drops that fall from it. In this case, it is difiicult to determine the volume of the liquid which has dripped out of the tube and, consequentl the original position of a given drop before the fractionation was started. Further, the resulting flow pattern tends to distort the zones. Still further, a gelatinous pellet of dense material often is formed at the bottom of the tube which interferes with, and may contarninate, the flow or" liquid from the tube.

Column chromatography is a process commonly used for the fractionation of smaller molecules than those fractionated by density gradient centrifugation. A hollow column is packed with a materifl through which different solutes, carried along by a moving solvent, will migrate at difierent speeds. When a homogeneous mixture of materials, dissolved in solvent, is in roduced at the top of the column, the materials migrate down the column at difierent rates. The dischar e from the bottom of the column will first contain the faster moving, and then the slower moving, material. At am given time, the dischar e from the column is usually of one species of material and/or pure solvent. A separation of the discharge flow into separate test tubes containing the different materials is accomplished by conventional means known as a fraction collector.

Apparatus made in accordance with this invention largely overcomes the difliculties heretofore attending the technique of density gradient fractionation and is also useful for fractionation by the column chromatography process. The apparatus makes it possible to measure continuously the optical density of the material being fractionated. This means that fine details in the light-absoroance profile, which are very important, will not be obliterated or rendered ambiguous, as is the case when samples are taken in batches from the centrifuge tube. -t is also possible to establish accurately where a given sample of the liquid was located in the tube before fractionation. The apparatus is automatic in operation and can be used with a recorder to provide a graph of optical density during the fractionating process.

An object of this invention is the provision of novel apparatus for fractionating chemical mixtures.

An object of this invention is the provision of means for fractionating liquids on the basis of optical density which apparatus includes automatic means for directing samples of the liquid having difierent optical densities into separate test tubes.

An object of this invention is the provision of a novel density gradient 'fractionator which includes means for automatically collecting the fractioned samples in separate test tubes with a minimum mixing of the flow stream thereby greatly improving the resolution of the analysis.

An object of this invention is the provision of continuous fractionating apparatus which includes .1 cans for continuously measuring the optical density of the liquid being fractionated.

An object of this invention is the provision of apparatus for the automatic removal of different density zone samples of a liquid from a density gradient tube comprising means for car the liquid to flow out of the gradient tube and through a delivery tube at a predetermined rate and with a minimum of turbulence, sensing means responsive to the changes in li ht absorption of the liquid as it passes to the delivery tube, and control means responsive to the sensing means for directing the discharge of fluid from the delivery tube into separate test tubes on the basis of its light-absorbing characteristic.

These and other objects and advantages of the invention will become apparent from the following description when taken with the accompanying drawings. It will be understood, however, that the drawings are for purposes of description and are not to be construed as defining the scope or spirit of th invention, reference being had for the latter purpose to the claims appended hereto.

In the drawings wherein like reference numerals denote like parts in the several views:

FIGURE 1 is a schematic diagram of apparatus made in accordance with this invention;

FEGURE 2 is a simplified circuit diagram of the differentiator, the two trigger circuits and the control relay;

FIGURE 3 is similar to PEGURE l and showing another embodhnent of the invention; and

FIGURE 4 is a diagrammatic representation of a novel light filter for use in the apparatus.

Reference now is made to FIGURE 1 wherein there is shown an optical r'low cell 1%), preferably made of Teflon plastic, which is chemically inert and withstands temperatures of 200 degrees C. Alternatively, for certain applications, the cell can be made of acrylic plastic which is resistant to aqueous solutions and vw'll withstand a temperature of 60 degrees C. in either case, the cell comprises a quartz window 12 (having a predetermined light path length),'a cap 11 (having 'a tapered, central aperture formed therein), and a bushing l3 (having an integral,

downwardly-depending annular ring portion of preferably triangular cross section). Means, not shown, in the drawing are provided for retaining the components in the illustrated, fixed, operative position while permitting easy disassembly thereof for cleaning. The window 12 is disposed in the optical beam generated by a lamp 14, which beam passes through a filter l and impinges upon a photoelectric cell "rd. Freierably, the lamp 14 is a low pressure mercury vapor lamp producing light having a predominant wave length of 254 millimicron and the filter 15 is esigned to transmit very pure monochromat c light, as will be described in detail hereinbelow.

A delivery tube 17 has one secured to the cap 11 (in alignment with the cap aperture) and the other end vertically spaced from a test tube 18 it may here be pointed out that a plurality of test tubes is carried by a conventional fraction collector which may consist of a turntable 19 having spaced discs provided with aligned holes for accommodating the test tubes in .a circular row. Such turntable is mounted for rotation by a turntable drive means 2% provided with a suitable indexing mechanism whereby energization of the drive means rotates the turntable an angular distance such that the adjacent test tube is brought under the end or" the delivery tube 37.

, The centrifuge tube Zl, preferably made'of thin plastic and carrying the density gradient column and the discrete zones of macromolecules after centrifugation, is positioned such that its open end fits tightly over the tapered end of the bushing 13. This tube is retained in the illustrated, operative position by any suitable means permitting easy placement and removal or" the tube, which means is represented in FIGURE 1 by the pivotallymounted lever 22 and tension spring 23. In accordance with one aspect of the invention, the hollow needle 24 of the syringe is caused to puncture the tube 21 proximate to its bottom, after the tube is pl ced in its operating position. Leakage around the puncture may be prevented by placing an annular foam rubber gasket 33 around the tube and puncturing through the gasket. The syringe carries a supply of liquid having a specific gravity exceeding that of the densest liquid contained within the tube, which dense liquid is forced into the bottom of the tube at a predetermined rate, by a conventional motor-driven screw and gear train generally identified by the reference numeral 25 in the drawing. The entry of the dense liquid into the tube gradually floats the density gradient column out of the tube, through the optical window 12 and through the delivery tube 17. a

As the liquid passes through the window 12,.the quantity of light striking the photocell 16 will vary in correspondence with the light-absorbing character of the liquid. The photocell is connected to a current amplifier 26 which, in turn, is connected to a diiiere'ntiator 27', the latter diflerentiating'the voltage applied thereto by the amplifier with respect to time. With a constant light energy level falling upon the photocell as, for example, when no liquid passes through the cell 12, the output of the amplifier remains constant whereby the voltage output of the differentiator 27 is zero. However, as the liquid in the tube'Zl is forced upwardly through the cell 12, a zone in the liquid, passing through the cell 12, will absorb a certain amount of the light energy whereby the light transmitted to the photocell is decreased, the light absorbing factor being related to V flov relay 2%. The relay 28 is energized through the diode 34 and the diode 35 prevents any electrical disturbance from reaching trigger circuit No. 2. The energization of relay causes actuation of the relay 29 having contacts for the mutua ly exclusive energization of the motor driven syringe 25 and the turn table drive 20. Specifically, as diagrammatically shown in the drawing, when the relay 29 is in the normal, deenergized condition, electric power is supplied to the motor driven syringe through the normally-closed relay contacts 30 and 31. When the operating coil of relay 2? is energized, the movable contact becomes disengaged from the stationary back contact El, thereby cutting off the power to the motor driven syringe, and engages the stationary front contact 32, thereby applying power to the turntable drive. The turntable drive 2% includes a drive motor and appropriate indexing mechanism whereby upon the momentary closure of the relay contacts 31 and 32, the table 19, carrying the test tubes, is rotated, or otherwise actuated, to bring the next test tube into position under the end of the delivery tube 1"]. It will be apparent, now, that the flow of the dense liquid into the bottom of the centrifuge tube 21 and, consequently, the flow of liquid out of the delivery tube 17, is stopped prior to movement of the turntable. In order to allow time for the movement of the turntable through a complete indexing cycle bef re further discharge of liquid from the delivery tube, the drop-out of the relay is delayed somewhat as by means of a latch-in device which will not release the relay until the fraction collector has moved and ithen'stop ed. Such means could be by electrical connections to the turntable drive 2% which would supply power to the coil of relay 2? wheh'the ttu'ntable drive 20 is operating.

When the light absorbance of the zone of liquid passing I through the optical cell reaches a maximum value, the voltage output of the amplifier remains constant whereby the output of the differentiator becomes zero. Tins causes trigger circuit No. 1 to return to its original state without any effect upon the relay 28 or trigger circuit No. 2 by reason of the polarity of the blocking diode 34.

As the particular zone of liquid under discussion passes through the cell 12, the li ht absorbance decreases, resulting in a decrease in the output voltage of the amplifier. This results in a positive voltage output from the differentiator which operates trigger circuit No. 2. However,

operation of trigger circuit No. 2 in the forward directron, does not energize the relay 28 by reason of the reversely-poled blocking diode-35 and, consequently, the

turntable remains stationary. 'When the light absorbing material has passed out of the cell 12, the light energy striking the photocell ceases to change whereby the output of the difierentiator drops to zero. This causes trigger circuit No. 2 to return to the original state and the resulting, reverse direction pulse causes momentary operation of the relay 28 and relay 29 thereby stopping the flow of liquid from the delivery tube and bringing the.

next test tube into position; Diode 34 prevents anyelectrical disturbance from reaching trigger circuit No. 1.

It will now be clear that the described apparatus operates on the light-absorbing characteristic of the liquid being lioatedupwardly through the cell 12 to automatically' collect in a single test tube a single zone of fluid contained in'the density gradient tube. The auto niatic operation of the apparatus is independent of the base line of the optical density before and after a particular zone of light-absorbing -material ,has passed through the optical cell. Further, successive peaks of light absorbance will result in the liquid zones producing such peaks being collected in separate test tubes, corresponding to each peak of the succession.

The apparatusshown in FIGURE 1 will function as described, provided the volumetric capacity. of each col 7 move the fraction collector at fixed intervals, such intervals corresponding to a volume of delivery less than the capacity of a collecting tube. However, some compensation must be made for the delivery lag between the optical cell 12 and the fraction collector. Specifically, the volume of the optical flow cell 12 and the delivery tube 17 causes the composition of the liquid reaching the collecting tube to lag behind that of the fiow cell. This will cause the fluid in the collecting tube to differ from that effecting the photocell. In actual practice, the delivery volume lag is of the order of 0.7 milliliter. While a lag of this magnitude usually is not serious, it can be a factor when the light absorption peaks are of short duration, as is often the case with density gradient fractionation. For this reason, it is preferable to include in the apparatus a time delay means 36 between the relay 29 and the relay 28. Such delay means may be of conventional design, such as, for example, a synchronous electric motor with associated, cam-operated switches which may be adjusted by the operator to delay operation of the turntable drive for a time period equal to the liquid transfer time between the optical cell and the collecting test tube.

A recorder 37 can be connected to the amplifier 26 for continuous measurement of the optical density of the liquid flowing through the optical cell 12. By properly coordinating the flow rate of the liquid with reference markings (which markings may be pre-printed on the recorder chart or automatically made thereon by conventional means each time the fraction collector moves), it is possible accurately to determine where a particular zone of liquid was located in the centrifuge tube 21 before fractionation and, also, in which of the several collecting tubes (on the fraction collector) the particular zone of liquid has been collected after the fractionation process.

Because of the uninterrupted and unobstructed vertical how of the liquid from the centrifuge tube, there is little,

if any, mixing of the flow stream. The mixing effect of the delivery tube 17 is minimized by inclining its discharge end upwardly at an angle, as shown in the drawing. The vertical density gradient column, with its inherent stability, is maintained and the discrete zones of liquid are not distorted by turbulence or the eflect of laminar fiow. The entire cross section of liquid tends to move at one discrete velocity.

Reference now is made to FIGURE 2, which is a simplified circuit diagram of the ditferentiator 27, the two trigger circuits and the relay 28. The diiferentiator circuit functions by operating upon the time derivative (slope or rate of change) of voltage which represents the optical density of the liquid flowing through the optical cell. Such voltage is obtained from the cathode of a conventional cathode follower section of the amplifier 26 (FIGURE 1) and is applied to capacitor 40. This capacitor, preferably a polystyrene dielectric capacitor, has a very high electrical leakage resistance. The output voltage from the capacitor, developed across one or both of the dropping resistors 41, 42 connected to the grid of the tube 43, approximates the time derivative of the optical density. A selector switch 44 having stationary contacts marked C, D and OFF is used to select the resistance values which determine the rate at which the time derivative is taken. A faster rate, with the smaller effective resistance connected in the circuit, preferably is used for density gradient fractionation, while the slower rate is used for column chromatography. This is because the light absorbance factor changes more slowly in column chromatography. The Zener diode 45 reversely conducts during any region of high negative voltage slope on the trailing edges of relatively large voltage peaks. This tends to reset the approximation of the time derivative to a lower absolute value so that it will have a near zero value shortly after a lar e voltage peak has passed. This decreases the delay in the automatic changing of collecting tubes after the passage of the voltage peak.

When the selector switch 44 is set to the OFF position, the grid of the tube 43 is connected to ground. This is done prior to adjusting of the derivative reference level by means of the rheostat 4a in the cathode circuit of the tube. The rheostat is set so that the anode potential is between the triggering levels of the double trigger circuits 47 and 48.

The double trigger circuits comprise the two tubes 50 and 51 (such as type 6BA7) with the input circuit connected to the anode of the dilferentiator tube 43 by the wire 52. it will be noted that the control grid 55, of the tube 51, is connected to the control grid 53 of the tube 5% through a resistor 54, whereby the control grid 55, of the tube 51, is biased at a higher potential than the grid 53. In consequence, the tube Sll triggers on a lower input voltage level than does the tube 59. The specific mechanism under which the two trigger circuits operate will be described in detail hereinbelow. For the present, it is pointed out that under quiescent conditions, the trigger tubes are in opposite stable states, with the anode of the tube 56 normally conducting and the anode of the tube 51 normally not conducting. A difference in potential between the two tubes causes the pilot lamp 5a to light.

When a light-absorbing zoneof liquid starts to pass through the optical cell, the time derivative of the optical density is positive and a positive voltage, which represents the positive time derivative, is applied to the grid of the diferentiator tube 43. This causes the anode potential of the tube to drop. When such voltage drops to the triggering level of the tube 51, its anode voltage suddenly drops as the anode triggers into conduction.

The relay 2-5 comprises a tube 57, such as a type 6C4, having the operating coil 53 of power relay 59 connected in the cathode circuit. The control grid of the tube 57 is connected to the anode circuit of the trigger tube 51 by a wire 69 and through the coupling diode 34- and the capacitor 61. When the trigger tube 51 conducts, its anode voltage suddenly drops with an attendent drop in the voltage applied to the control grid of the tube 57. This causes a momentary deenergization of the operating coil 58 of the relay 59 resulting in a momentary closure of the relay contacts 62, 63. Such contact closure results in the operation of the relay 29, see FIGURE 1, to stop the motor-driven syringe 25 and to effect operation of the turntable drive 24? to bring the next adjacent collecting tube into registry under the delivery tube 17.

As the voltage peak reaches its maximum value, its time derivative drops to zero and the trigger tube 51 reverts to its original state. The sudden rise in the anode potential, at this time, causes no circuit disturbance because the trigger tube is cut off from the rest of the circuit by the blocking action of the coupling diode 34.

As the voltage peak decreases back to zero, its time derivative is negative and the anode potential of the differentiator tube 43 rises. This causes a tri gering of the trigger tube 59 and its anode current cut off. The anode potential rises sharply but the effect of the voltage rise is blocked from the relay 28 by the coupling diode 35. After the voltage peak has passed, the time derivative drops to Zero and the trigger tube 5t) reverts to its original state. This sudden lowering of the anode voltage of the tube 59 becomes effective on the grid of the relay tube 57 thereby again causing a momentary closure of the relay contacts 62, 63 and eifecting an indexing operation of the fraction collector to bring the next collecting test tube under the end of the delivery tube.

The action of the trigger circuit will now be explained in somewhat more'detail. Assume that the anode current of the trigger tube 51 is cut off because the potential on the secondary control grid 65 is considerably lower than the cathode potential. In this condition, almost all of the cathode current will flow in the screen grid 66. If the control grid 55 is made more negative, the cathode and screen current will drop and the screen grid voltage 7 possible.

7 will rise. Such voltage rise will be transmitted by the voltage-dropping Zener diode 57 to the secondary control control grid 65 by the Zener diode 67, whereby the grid 65' becomes more negative and repels more of the cathode current towards the screen grid 66. This, then, tends to increase the screen current with an attendant drop in the screen potential. Such action is cumulative and the trigger tube sharply reverts to its original state with a substantially zero anode current and a high anode potential.

Reference now is made to FIGURE 3 showing the use of the apparatus in connection with the column chromatography process, like parts in the FIGURE 1 and FIG- URE 3 arrangments being identified by like reference numerals. The hollow, vertically-disposed column 7'0, packed with appropriate material, is positioned over the tapered end of the bushing 13'. Alternatively, the column can be connected to the optical cell 12 by means of appropriate tubing. The homogeneous mixture of materials, dissolved in a solvent, is introduced onto the top of the column and the materials will migrate down the column at different rates, depending on time or the external changing of the solvent as it moves down through the column. The material discharged from the column will first contain the faster moving materials and then the slower. Thus, the liquid flowin' through the optical cell 12 and the delivery tube 17', at any particular time, is usually of one species of material and/ or pure solvents. Separation of the discharge flow into separate test tubes containing the different materials is accomplished automatically by the apparatus which has been described hereinabove in detail with reference to FIGURE 1.

Reference now is made to FIGURE 4, which illustrates an improved light filter generally identified by the numeral 15'. The three elements which make up the filter are supported in fixed position by a suitable housing 72. The element 73 is made of a material which is transparent to the entering ight of desired wave length. The element 74 is a material which fluoresces under the influence of the entering light of desired wave length and the element 75' is made of a material which transmits light of the wave length emitted by the fluorescence of element 74. It is desirable, but not necessary, that the element 73 have a high light absorbance factor at wave lengths where the element 75 transmits light. .t is also desirable that the photocell 16, used with the particular filter, be responsive only to light or" a wave length corresponding to that of the fluorescence of the element 74. The filter, when used with a'spectral lamp, makes it possible to obtain a much narrower effective wavelength than can be obtained by conventional absorption or interference filters. For most applicationsinvolving the measurement of light transmittance or reflectance of specimens, it is necessary that the effective bandwidth of the illumination be as narrow as This desirable result can be achieved in the present filterbe cause the element 73 can be made of material selected to have a cut oil to wave lengths shorter than the spectral line of interest (supplied by lamp 14) and the element 74 can be made of material selected to have a cut wave length for excitation of fluorescence at a a. slightly longer 'Wave length than the spectral line of interest. As an'illustratiom'the following arrangement has been found to provide a very narrow, efiective bandwidth-with a stray light factor of less than one thousandth or the magnitude of desired wave length.

"The lamp l used is a low pressure, 'mercury vapor a light of 254 millimicron wave length. The element 73 chosen to fluoresce green when excited by wave lengths shorter than 280 millimicrons. The element is green colored glass and the photocell 16 is green-sensitive. Such filter arrangement will respond only-"to light in the range of 240 millimicrons (the cut off of element 73) to 280 millimicrons (the cut oil of element 73). Within this region, the exciting lamp has only one significant wave length, namely, 254 millimicrons. The entire system, as a whole, responds only to a wave length of 254 millimicrons. Contrastingly, if an interference or absorbance filter were used instead, stray light would exist, especially of a wave length of 313 millimicrons.

Having described my invention, those skilled in this art will be able to change and modify theillustrated embodiments to meet specific conditions or applications. As an example, in the FIGURE 1 embodiment, it is possible to inject the dense solution into the bottom of the centrifuge tube through a hollow needle extending down into the tube from its top end. Also, the motordriven syringe can be replaced by various other arrangements for forcing the dense liquid into the centrifuge tube at a predetermined, constant rate. Specifically, a manual hand crank drive can be'uscd for advancing the syringe, with the crank cooperating with a suitably calibrated dial for determining the amount of liquid which has been delivered into the centrifuge tube. Thislatter arrangement can also be used in determining the volume of liquid between the optical cell and the end' of the delivery tube. Additionally, cam-operated switches can be operated in accordance with rotation of the hand crank or motor drive to cause a positional change in the col- 7 lecting test tubes.

Further, the photocell, etc., can be replaced with conventional sensing elements for other physical or chemical factors than light absorbance, such as conductivity, pH, redox potential, fluorescence, etc. This will allow automatic fractionation on the basis of these other properties of the flow stream.

The electrical output of this fractionator, normally used to cause actuation of the fraction collector, can be used for other purposes. An example is to start and stop an integrator to determine the area under each peak on the recording chart. 7

It is intended that these and other changes and modi: 'fications can be made without departing from the spirit and scope of the invention as recited in the following claims.

l claim:

1. Apparatus for fractionating a vertical densitygradient tube, which apparatus comprises an optical cell disposed over the density-gradient tube, means for in-' jecting a heavy liquid into the bottom of the densitygradient tube thereby to float the contained liquid out of the tube and through the optical cell, sensing means providing a signal which varies with the transmission of light through the optical cell, and means responsive 'to the said signal for producing a corresponding visual indication. V A

. 2. The invention as recited in claim 1, including a fraction collector for receivingliquid discharged from the optical cell. a

3. Apparatus for fractionating a vertical densitygradient tube, which apparatus comprises an optical cell disposed over the density-gradient tube, means for in-' jecting 'a heavy liquid. into the bottom of the densitygradient tube'therebyto float the contained liquid out of the tube and'through the optical cell, sensing means,

providing a signal which varies with the transmission of' light through the optical cell, and a recorder responsynchronized with the flow rate of the heavy liquid injected into the density-gradient tube.

4. The invention as recited in claim 3, including a traction collector having a plurality of collecting tubes, power means for moving the fraction collector to successively place the collecting tubes in position to receive liquid discharged from the optical cell, and means stopping the flow of heavy liquid into the density-gradient tube during movement of the fraction collector.

5. The invention as recited in claim 4, including means delaying the operation of the power means for a time period corresponding to the liquid transfer time between the optical cell and a collecting tube positioned to receive liquid therefrom.

6. The invention as recited in claim 1, wherein the said sensing means comprises a photoelectric cell positioned at one side of the optical cell and a mono-chromatic light passing through the optical cell and striking the photoelectric cell.

References Cited in the file of this patent UNITED STATES PATENTS 2,645,379 Audia July 14, 1953 2,708,389 Kavanagh May 17, 1955 2,741,157 Goethert Apr. 10, 1956 2,880,764 Pelavin Apr. 7, 1959 2,954,045 Leek Sept. 27, 1960 3,004,567 Snow et a1 Oct. 17, 1961 3,036,736 Murphy et a1. May 29, 1962 

1. APPARATUS FOR FRACTIONATING A VERTICAL DENSITYGRADIENT TUBE, WHICH APPARATUS COMPRISES AN OPTICAL CELL DISPOSED OVER THE DENSITY-GRADIENT TUBE, MEANS FOR INJECTING A HEAVY LIQUID INTO THE BOTTOM OF THE DENSITYGRADIENT TUBE THEREBY TO FLOAT THE CONTAINED LIQUID OUT OF THE TUBE AND THROUGH THE OPTICAL CELL, SENSING MEANS PROVIDING A SIGNAL WHICH VARIES WITH THE TRANSMISSION OF LIGHT THROUGH THE OPTICAL CELL, AND MEANS RESPONSIVE TO THE SAID SIGNAL FOR PRODUCING A CORRESPONDING VISUAL INDICATION. 